Hybrid cell lines producing monoclonal antibodies directed against neurotransmitter degrading enzymes

ABSTRACT

A continuous hybrid cell line which produces monoclonal antibody directed against the neurotransmitter degrading enzyme, monoamine oxidase B (MAO B), has been developed. The hybrid cell line was established by fusing MAO B primed, differentiated lymphoid cells with myeloma cells. Resulting fused cells were isolated, cloned and characterized as to antibody specificity against antigenic determinants of MAO B. Monoclonal antibody having specificity for MAO B and no cross reactivity with MAO A was selected and implemented in a radioimmunoassay technique for the selective measurement of MAO B concentration independent of its catalytic activity.

BACKGROUND OF THE INVENTION

The present invention relates to hybrid cell lines capable ofcontinuously producing monoclonal antibody specific for neurotransmitterdegrading enzymes and to immunoassay methods using the monoclonalantibodies.

In recent years, the capability to produce monoclonal antibodiesspecific to immunogenic determinants of bacterial cells, viruses, tissuecomponents and proteins has provided a new spectrum of diagnostic andimmunotherapeutic agents.

The major intracellular enzyme responsible for the metabolic degradationof catecholamines in mammals is monoamine oxidase (MAO). This enzyme,which is located in the outer mitochondrial membrane and exists in twoforms (A and B), plays an important role in the nervous system. It isalso believed to function in regulating the level of pressor amines,such as phenylethylamine, in the circulation and in non-neuronaltissues. MAO catalyzes the oxidation of amines to their correspondingaldehydes, which are rapidly metabolized, usually by oxidation to anacid.

Because MAO contributes to determining the concentration of themonoamine neurotransmitters, the activity of this enzyme has beenstudied in patients with a wide variety of neurological and psychiatricdisorders. The B form of the enzyme is found in most human tissues (withthe exception of placenta, where MAO A is the predominant form) and itspresence in blood platelets provides a convenient tissue for study ofits activity in pathological states. In fact, platelet MAO B level hasbeen found to be reduced in chronic schizophrenic patients compared tonormals in many studies, a finding which has led investigators to focuson MAO as a possible biological marker in schizophrenia. Low plateletMAO activity, however, has also been reported in bipolar affectiveillness, alcoholism, Down's Syndrome, iron deficiency anemia, essentialhypertension, migraine, and juvenile diabetes. In addition, low MAOactivity has been associated with suicide, sensation seeking and morefrequent psychiatric counseling.

As indicated above there are two types of MAO, A and B, each of whichhas distinct catalytic properties and is expressed in variousproportions in different tissues.

MAO A is selectively inhibited by low concentration of the irreversibleactive site inhibitor clorgyline and preferentially oxidizes lowconcentrations of 5-hydroxytryptamine.

MAO B is selectively inhibited by low concentrations of deprenyl andpargyline and preferentially oxidizes low concentrations ofphenylethylamine, and benzylamine.

Although the distinguishing catalytic activities of MAO A and B havebeen evaluated to some degree, there is little known of the structureand molecular properties of MAO A and B. MAO B, the most extensivelystudied of the two enzymes, has been characterized as having molecularweight of approximately 120,000 and consists of two subunits ofindistinguishable molecular weight. One subunit has an essentialmolecule of covalently bound flavin adenine dinucleotide. Moreover, MAOmay have carbohydrate residues added to its polypeptides and its lipidmicroenvironment is thought to contribute significantly to its catalyticactivity.

Since the measured level of MAO activity is a reflection of severalfactors (primary structure, covalent modification, andmicroenvironment), it is important to examine independently MAO activityand MAO concentration. Such a dissection of activity and concentration,along with sequencing information, should clarify the fundamentalstructure and function of this enzyme and suggest how alterations inthem, if they exist, could lead to abnormal MAO activity in psychiatricdisorders.

Several assays of MAO catalytic activity, including the most commonlyused radioenzymatic assay, have been developed, but only one method hasbeen available heretofore to measure the concentration of MAO protein.

The assay of active MAO concentration involves the titration of enzymeactivity with known amounts of ³ H-pargyline. This inhibitor reactsspecifically and irreversibly on a mole to mole ratio with thecovalently bound FAD of MAO B. Knowing the specific activity of the ³H-pargyline used in the reaction permits determination of the nmoles ofpargyline bound in the preparation, which in turn is equal to the nmolesof MAO in the sample. Since the method measures only catalyticallyactive MAO, molecules are not measured which are physiologicallyinactive or which are inactivated during extraction or non-specificallyby the incubation conditions used for ³ H-pargyline binding.

An alternative method for determining MAO concentrations involves theuse of an antibody which binds MAO B specifically. Heretofore, thereagent antibody has been an antiserum prepared from rabbits immunizedwith highly purified MAO B. Although this reagent was used in rocketimmunoelectrophoresis to determine the concentration of MAO in plateletsamples from patients, the antiserum has had limited application becauseof variability in animal response to the MAO antigen and the relativelylow titers obtained. Such difficulties are commonly encountered inproducing specific antisera.

Accordingly, in efforts to determine accurately the molecularconcentration of MAO or other neurotransmitter degrading enzymes, thereexists a need for unlimited quantities of an unvarying antibody reagentwhich recognizes a single antigenic determinant of the enzymes. Suchantibody reagents are provided by this invention.

SUMMARY OF THE INVENTION

In accordance with the present invention, continuous monoclonal hybridcell lines are established which elaborate and secrete highly specificand homogenous monoclonal antibody directed against a neurotransmitterdegrading enzyme.

In its broadest aspect, the invention involves first immunizing ananimal in vivo or antibody producing cells in vitro to neurotransmitterdegrading enzymes. Neurotransmitter degrading enzymes include generallymonoamine and catecholamine degrading enzymes such as monoamineoxidases, catechol O-methyltransferase and benzylamine oxidase. Afterimmunization, the primed lymphocytes are recovered and fused withmyeloma, plasmacytoma, or hybridoma cells to form somatic cell hybrids.

The cell hybrids are cultured, selected, and propagated in tissueculture or in vivo in ascites fluid. Thereafter the hybrid cell linesare capable of continuously producing monoclonal antibodies to theselected immunizing antigens.

Monoclonal antibodies derived and collected from an isolated hybrid cellline can be implemented in immunoassay techniques for the detection of aspecific antigen in tissue samples.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

The following discussion is in terms of the preferred embodiments ofthis invention, which represent the best mode known to the Applicants atthe time of this application.

In accordance with this invention, monoclonal antibodies directedagainst the neurotransmitter degrading enzyme, MAO B, were isolated fromcontinuous hybrid cell lines formed by the fusion of antigen-primedimmune lymphocytes with myeloma cells.

Monoclonal antibodies are highly specific, being directed against asingle antigen only. Furthermore, in contrast to conventional antibodypreparations which typically include different antibodies directedagainst different sets of determinants on the same antigen, monoclonalantibodies are directed only against a single determinant on theantigen. Monoclonal antibodies are useful to improve the selectivity andspecificity of diagnostic and analytical assay methods usingantigen-antibody binding. A second advantage of monoclonal antibodies isthat they are synthesized in pure form by the hybridoma culture,uncontaminated by other immunoglobulins. Monoclonal antibodies may beprepared from supernatants of cultured hybridoma cells or from ascitesinduced by intraperitoneal inoculation of hybridoma cells into mice.

The hybridoma technique described originally by Kohler and Milstein,Eur. J. Immunol. 6:511-519 (1976) has been widely applied to producehybrid cell lines that secrete high levels of monoclonal antibodiesagainst many specific antigens. In accordance with the production ofthis invention, MAO was partially purified from physiological tissuesamples by ammonium sulfate precipitation and DEAE sepharosechromatgraphy. After partial purification the enzyme was labeled with ³H-pargyline (this irreversible inhibitor binds only to MAO in thesepreparations) to provide a convenient method for following the enzymeduring subsequent purification steps. MAO was further purified byisoelectric focusing or chromatofocusing, and a preparation in which MAOrepresented 20 to 30% of the total protein was used to immunize a BALB/cmouse.

Alternatively normal and immune differentiated lymphocytes capable ofproducing antibody can be isolated from test animals and cultured invitro to generate cells appropriate for producing lymphocyte hybridomas,for example such methods as in vitro stimulation of lymphocytes withmitogens and/or antigens as described by Robertson et al, Microbiology1980 pp. 181-185 (1980) and Kettman et al, J. Immunol. Methods39:203-222 (1980) or the splenic fragment culture method as described byPress et al, Eur. J. Immunol. 4:155-159 (1974).

The route and schedule of immunization of the host animal or culturedantibody producing cells therefrom are generally in keeping withestablished and conventional techniques for antibody stimulation andproduction. Applicants have employed mice as the test model although itis contemplated that any mammalian subject including human subjects orantibody producing cells therefrom can be manipulated according to theprocesses of this invention to serve as the basis for production ofmammalian, including human, hybrid cell lines.

After immunization, immune lymphoid cells are fused with myeloma,plasmacytoma, or hybridoma cells (hereinafter referred to collectivelyas myeloma cells) to generate a hybrid cell line which can be cultivatedand subcultivated indefinitely, to produce large quantities ofmonoclonal antibodies. For purposes of this invention, the immunelymphoid cells selected for fusion are lymphocytes and their normaldifferentiated progeny, taken either from lymph node tissue or spleentissue from immunized animals. Applicants prefer to employ immune spleencells, since they offer a more concentrated and convenient source ofantibody producing cells with respect to the mouse system. The myelomacells provide the basis for continuous propagation of the fused hybrid.Myeloma cells are tumor cells derived from plasma cells which showpreference for bone marrow. Plasmacytoma cells are neoplastic cellsderived from plasma cells. In particular, Applicants prefer to uselymphocyte hybridoma cells which secrete no immunoglobulin. Lymphocytehybridoma cells are cells generated by the fusion of myeloma orplasmacytoma cells with normal differentiated lymphoid cells. Myeloma,plasmacytoma, and hybridomas can be selected to be devoid ofimmunoglobulin synthesis.

The particular species of animal from which the myeloma and immunizedantibody producing cells are derived are not critical, in that it ispossible to fuse cells of one species with another. However, it ispreferred that the source of immunized antibody producing cells andmyeloma be from the same species.

Generally the fusion techniques employed are according to the proceduresset out by Kohler et al, Eur. J. Immunol. 6:11-19 (1976) and Kennett etal, Lymphocyte Hybridomas--Current Topics In Microbiology and Immunology81:77-91 (1978) Springer-Verlag, New York. Fusion is generallyaccomplished by adding a suspension of antibody producing cells to themyeloma cells in growth medium and centrifuging in the presence ofpolyethylene glycol to form a pellet.

Products of the hybridization embodied by this invention were screenedfirst for their ability to recognize any antigen in the chromatofocusedpreparation and then for their ability to indirectly immunoprecipitate ³H-pargyline labeled MAO B. The fusion produced approximately 300 clones.Thirty-four of the clones secreted antibody which bound to microtiterwells coated with the chromatofocused MAO and could be detected by aperoxidase-linked immunosorbent assay (ELISA). One clone produced anantibody, MAO-1C2, which recognized both pargyline inactivated andcatalytically active MAO B and failed to crossreact with MAO A.Following subcloning, cells secreting MAO-1C2 were injectedintraperitoneally into BALB/c mice to generate ascites fluids containinglarge amounts of MAO-1C2.

Alternatively, the hybrid cell lines can be continued to be cultured invitro on cell culture media.

Moreover the hybrid cell lines can be stored and preserved in any of anumber of conventional ways, including freezing and storage under liquidnitrogen. Frozen cell lines can be revived and cultured indefinitelywith resumed synthesis and secretion of monoclonal antibody. Thesecreted antibody is recovered from tissue culture supernatant byconventional precipitation, ion exchange, affinity chromatography, orthe like. The recovered antibody can be frozen and stored underrefrigeration at -80° C. for at least one year without significant lossof activity.

The availability of large amounts (gram quantities) of the wellcharacterized monoclonal antibody MAO-1C2 as provided by this inventionmakes it possible to measure MAO protein concentration in extracts ofplatelets from patients with neurological and psychiatric disorders forcomparison with normal controls. Further in accordance with thisinvention, Applicants have developed a radioimmunoassay which is ahighly reliable and specific method for determining concentration of MAOprotein independent of its catalytic activity. By determining the amountof MAO protein and the catalytic activity of the enzyme in the sample,the molecular activity of MAO B in crude extracts of human platelets canbe determined. In clinical studies of patients with schizophrenic,affective, and other psychiatric disorders, this methodology will permitassessment of abnormalities due primarily to altered molecular activityor enzyme concentration.

The following examples are offered to illustrate a particular embodimentof the invention but they are not intended to limit it.

A. Preparation of Antigens

The MAO preparation used for immunization was purified from outdatedhuman blood platelets elaborating predominantly B type enzyme.

Platelet rich plasma (PRP) was obtained from The University of TexasMedical Branch Blood Bank immediately after the platelets becameout-dated (72 hours after blood drawing). The PRP was stored in acold-room overnight. In each MAO preparation, a batch of 25 units (65ml/unit) was used.

Pooled PRP was centrifuged at 600×g for 3 min to remove contaminatingred blood cells and lymphocytes. The supernatant was centrifuged at2,500×g for 20 min and the platelets collected. The platelets werewashed by dispersing in 0.9% saline-5 mM EDTA (saline-EDTA), pH 7.4, andthen centrifuged at 2,500×g for 20 min. After two more washings withsaline-EDTA solution, the washed platelets were suspended in colddistilled water to a final protein concentration of 5 mg/ml. Thesuspension was frozen at -20° C. overnight, thawed, and centrifuged at35,000×g for 60 min. The pellet was suspended in 50 mM potassiumphosphate buffer, pH 8.0, containing 0.1% Triton X-100 (freshlyprepared). After the suspension was stirred for 60 min at 4° C., it wascentrifuged at 35,000×g for 30 min. The pellet was resuspended in 50 mMpotassium phosphate buffer, pH 8.0, containing 0.5% Triton X-100,stirred for 60 min at 4° C. and centrifuged at 150,000×g for 60 min. Thesupernatant containing solubilized MAO was dialyzed against 3×6,000 mlof 10 mM potassium phosphate buffer, pH 8.0, for 36-40 h.

Then the dialyzed-extracted MAO was fractionated on a DEAE-Sephacel(Pharmacia) column (2.6×40 cm) which had been previously equilibratedwith 10 mM potassium phosphate buffer, pH 8.0. The column was developedby stepwise elution with 10 mM and 100 mM potassium phosphate buffer, pH8.0, and MAO was eluted with 100 mM potassium phosphate buffer, pH 8.0,containing 0.25% Triton X-100. The fractions containing high MAOactivity were pooled together and the active protein was precipitated byadding solid (NH₄)₂ SO₄ to 50% saturation. The mixture was centrifugedat 30,000×g for 60 min and the precipitate which floated on the surfacewas collected. The precipitate was dissolved in 50 mM potassiumphosphate, pH 8.0, containing 1% octylglucoside.

To facilitate monitoring the enzyme during subsequent purification, MAOin the combined, active fractions from the DEAE column was labeled bytreatment with 0.06 μM ³ H-pargyline (New England Nuclear, sp. act., 50Ci/mmol) for 30 min at 37° C. and then dialyzed against 3×6,000 ml of0.025 M Tris-acetate buffer, pH 7.4, or 36-40 hrs. The final specifityactivity of the MAO was 96,000 cpm/μg MAO protein. Estimates ofquantities of ³ H-pargyline labeled MAO are based on ³ H cpm, assumingthe specific activity reported above, and assuming a molecular weight of120,000 for MAO. Labeling of the enzyme with ³ H-pargyline under theseconditions resulted in >90% inhibition of the enzyme. The ³ H-pargylinelabeled MAO was further fractionated on a Polybuffer Exchanger 74(Pharmacia) chromatofocusing column (0.9×27 cm) according to theinstructions from Pharmacia Fine Chemicals. The pH gradient wasdeveloped by elution with 200 ml of eight times-diluted Polybuffer 74(Pharmacia), pH 4.0, (adjusted with 1 M HCl) containing 1%octylglucoside. The 2.8 ml fractions were collected and were assayed forabsorption at 280 nm, pH, and MAO activity. The fractions showing highMAO radio-activity near pH 5.3 were pooled together and the labeledprotein fraction was precipitated by adding solid (NH₄)₂ SO₄ to 80%saturation. The floating precipitate collected after centrifugation at30,000×g for 20 min was washed once with 80% saturated solution of(NH₄)₂ SO₄ in 50 mM potassium phosphate buffer, pH 7.4. The washedprecipitate was dissolved in 50 mM potassium phosphate buffer, pH 8.0,containing 1% octylglucoside and dialyzed against 6000 ml of 10 mMpotassium phosphate buffer, 7.4, for 24 h.

The chromatofocused preparations had specific radioactivity,20,000-30,000 cpm/μg total protein, and it was estimated that MAOconstituted 20-30% of the total protein. Virtually all the ³ H in theDEAE-purified and chromatofocused MAO samples migrated as a single peakat a molecular weight of ca. 59,000, close to the molecular weightreported for the FAD-containing subunit of human MAO B.

B. Immunization of Animals

A BALB/c mouse was immunized by two intraperitoneal (i.p.) injections of10 μg of ³ H-pargyline labeled platelet MAO (days 1 and 7) contained in0.1 ml of sterile PBS and emulsified in an equal volume of complete(first injection) or incomplete (second injection) Freund's adjuvant.The animal was boosted by i.p. injection of 10 μg of ³ H-pargylinelabeled MAO (chromatofocused material) in saline on days 54, 55, and 56,and the fusion performed on day 57.

C. Construction of Hybridomas

Hybridomas were produced by fusing spleen cells prepared on day 58 (oneday following the last boost) to P3/X63 Ag8 myeloma cells with 40%polyethylene glycol 1,000 (Sigma Chemical Co.). P3/X63 Ag8 cells wereobtained from Dr. Roger Kennett, Department of Human Genetics,University of Pennsylvania School of Medicine.

The hybridoma fusion techniques were performed according to theprocedure of Kennett et al, Lymphocyte Hybridomas--Current Topics inMicrobiology and Immunology, Vol. 81, pp. 77-91 (1978) Springer-Verlag,New York.

Spleens were removed aseptically from immunized mice and teased apartgently with forceps to prepare a single cell suspension in Dulbecco'sModified Eagle's Medium (DMEM). P3/X63 Ag8 cells were harvested in thelogarithmic phase of growth and both cell types were collected bycentrifugation at 270×g for 10 minutes at room temperature and washedthree times with DMEM.

Approximately 10⁸ spleen cells were mixed together with cells in a 50 mlconical tube at a ratio of 10 viable spleen cells per viable P3/X63 Ag8cell and the resultant cell suspension was collected in a pellet bycentrifugation at 270×g for 10 minutes. The supernatant medium wasremoved and the tube containing the cell pellet was placed in a 37° C.water bath. A 0.2 ml portion of a warm (37° C.) 35% (wt/vol) solution ofpolyethylene glycol in DMEM was added to the cell pellet which was thengently mixed. The cell suspension was incubated at 37° C. for 3 minutesand was then collected by centrifugation at 270×g for 6 minutes.

Warm DMEM (5 ml) was gently dropped onto the cell pellet and the cellswere suspended by gentle agitation. An additional 5 ml of warm DMEM wasthen added and the cells were collected by centrifugation. This finalcell pellet was resuspended in 25-30 ml HY medium (see Kennett et al,supra at p. 78) and dispensed in 50 μl portions containing 1.9×10⁵ cellseach into 512 microtiter plate wells (Costar Plastics). Plates wereincubated at 37° C. in 10% CO₂ /air. Aminopterin (final concentrations0.018 mg/ml) was added the day after fusion. Cells were fed on days 7and 14 after fusion with HY medium lacking aminopterin.

D. Screening Hybridomas for Specific Antibodies

Cell culture supernates were screened initially for antibody capable ofbinding antigen in chromatofocused MAO which would coat microtiterplates and which could be detected by a peroxidase-linked immunosorbentassay (ELISA).

³ H-pargyline labeled MAO (chromatofocused material) was incubated for 4hr at 37° C. in Cooke polystyrene microtiter plates (1 μg/ml MAOportein) in borate-saline buffer (per liter, 6.2 g H₃ BO₃, 9.5 g Na₂ B₄O₇.10H₂ O, 9.0 g NaCl, pH 8.2). Conditioned media from wells containinggrowing clones were diluted 1/10 with PBS plus 0.05% Tween 20 (SigmaChemical Co.) and incubated in the washed wells for 4 hr at 23° C. Boundmouse immunoglobulin was detected colorimetrically after a further 4 hrincubation of the washed wells with peroxidase-conjugated sheepantimouse IgG (heavy plus light chain, 1/1000 dilution from CappelLaboratories, Cochranville, Ill.). The peroxidase reaction mix (150μl/well) contained per 20 ml of citric acid buffer, pH 5; 8 mgo-phenylenediamine, Sigma Chemical Co.; and 4 μl of 30% hydrogenperoxide. Reactions were stopped after 2-4 min with 50 μl of 4 Msulfuric acid. Absorbance data was quantitated using an Automated ELISAreader, model MR 580 (Dynatech Laboratories).

Most cell culture supernatants gave a low level of peroxidase activityindistinguishable from control medium (fresh culture medium or mediumconditioned by P3/X63 Ag8 cells), but 31 gave color reactions judged tobe significantly above background. After expansion of the cellpopulations to ca. 5×10⁶ cells for freezing in liquid nitrogen, 14primary clones tested were strongly positive by ELISA assay. Thetitration curves, of the selected 14 primary clones differed markedly inshape from clone to clone, suggesting that the conditioned mediacontained diverse antibodies which apparently recognized a variety ofantigenic determinants.

In order to determine whether any of the ELISA-positive antibodies couldbind MAO, conditioned media was screened by indirectimmunoprecipitation.

Conditioned media from each of the ELISA-positive hybridomas werediluted 1/10 with NET buffer (0.15 M NaCl, 5 mM EDTA, 50 mM Tris, 0.02%sodium azide, pH 7.4) containing 0.1% bovine serium albumin and 0.05%NP-40 (Particle Data Corporation), and mixed with 20-40 ng of ³H-pargyline labeled MAO contained in 50 μl of the same buffer. Thesesamples were incubated in 96-well polystyrene microtiter plates (Cooke)at 23° C. for 1 hr in a rotary shaker (50 μl total volume per well).Rabbit antimouse IgG (heavy plus light chain; Cappel Laboratories,Cochranville, Ill.) was added (equivalent to 8 μg of specific antibody),and the incubation continued for 1 hr. Heat-killed fixed Staphylococcusaureus Cowan I (Pansorbin A; Calbiochem) was then added (50 μl of a 10%suspension), and the incubation continued for 15 min. The plate was thencentrifuged for 10 min at 1700 rpm (5° C.) in a Cooke microplate carrier(Dynatech Laboratories, Alexandria, VA.) in a IEC refrigeratedcentrifuge (model PR-6000).

The resulting supernatants were then assayed for catalytically inactive³ H-pargyline labeled MAO by counting 50 μl samples dried onto 2.5 cmglass fiber filters (Reeve Angel) in toluene-based scintillation fluid(containing per L of toluene, 4g of PPO, and 0.05 g of dimethyl POPOP)or suspended in 2 ml PCS scintillation fluid (Amersham Corp. ArlingtonHeights, Ill.). Counting efficiency for ³ H was 20% on dried filters or25% in PCS when counted in a Packard-Tri-carb Liquid ScintillationSpectrometer.

Of twelve conditioned media tested, all left more than 70% of the ³H-pargyline labeled MAO in solution when S. aureus cells were used assecondary reagent. Furthermore, more than 68% of the label was left insolution when medium from eleven of the twelve primary clones (all butclone 1C2) and both S. aureus cells and rabbit anti-mouse IgG werepresent in the assay suggesting that at most a small proportion of the ³H-pargyline labeled MAO was immunoprecipitated by supernatants fromthese clones. However, one conditioned medium, 1C2, immunoprecipitatedall but 15.6% of the label from the supernatant, when both secondaryreagents were present. Pelleted bacteria from experiments involving 1C2were washed by centrifugation. Bound radioactivity determination showedthat a combination of (a) conditioned medium from primary clone 1C2, (b)rabbit antimouse IgG, and (c) S. aureus cells immunoprecipitated 20-foldmore ³ H-pargyline labeled MAO than any other sample. Under appropriateconditions, antibody from hybridoma 1C2 will precipitate at least 95% of³ H-pargyline labeled MAO B. Because of its ability to immunoprecipitateMAO in indirect immunoprecipitation assays, antibody from well 1C2 wasstudied further.

Hybridoma MAO-1C2 was grown continuously for 3 months (about 90generations) without apparent loss of specific antibody secretion. Inthe meantime, eleven subclones were isolated, and supernatants from 6 of6 subclones tested were found to secrete MAO-binding antibody as judgedby their ability to immunoprecipitate ³ H-pargyline labeled MAO inindirect immunoprecipitation tests. One of these subclones, termedMAO-1C2 #8, was injected (3×10⁶ -10⁷ cells) into pristane-primed mice togenerate ascites fluid. Indirect immunoprecipitation assays indicated 1ml of ascites fluid could bind the equivalent of 14.1 mg of MAO.

MAO-1C2 #8 was resubcloned after an additional 3 mo in culture, and 7 of9 secondary subclones secreted anti-MAO antibody. Therefore, specificantibody secretion by this hybridoma appears to be reasonablygenetically stable.

Since MAO-1C2 was elicited to pargyline-inhibited enzyme and detected byits ability to bind the same material, it was important to determinewhether it could bind catalytically active enzyme. The antibody wasfirst tested for its ability to inhibit MAO B activity in extracts ofhuman liver mitochondria as assayed by benzylamine oxidation by theradiometric technique of Wurtman and Axelrod, Biochem. Pharmacol.12:1439-1440 (1963). An extract of human liver mitochondria hadsignificant activity (5.15±0.057), and the addition of MAO-1C2 scarcelyreduced this at all (4.93±0.034), indicating that the antibody did notinhibit the enzyme, nor did MAO-1C2 alone immunoprecipitate enzymeactivity. When MAO-1C2 and rabbit antimouse IgG were both added, aprecipitate was formed which could be pelleted at low speed. Assays ofthe MAO activity in the supernatant and resuspended pellet of thissample showed that most of the activity was in the pellet (3.91 units inthe pellet, 5.06 units in the pellet and supernatant combined). Sincethe activity recovered (5.06 units) was very close to the amount ofactivity added to the well (5.15 units), it is concluded that MAO-1C2plus antimouse IgG precipitated the enzyme without inhibiting it.Furthermore, the addition of S. aureus cells to the mixture of enzymeplus MAO-1C2 plus antimouse IgG did not give any further inhibition orprecipitation of enzyme activity.

Further indirect immunoprecipitation tests using catalytically activeenzyme indicated that MAO-1C2 did not distinguish between catalyticallyactive and ³ H-pargyline labeled MAO. Moreover, indirectimmunoprecipitation tests involving crude placental mitochondrialextract indicated MAO-1C2 monoclonal antibody did not recognize MAO A.

Furthermore, the monoclonal antibody MAO-1C2, derived ultimately frommice immunized with human MAO-B priming, does not bind either mouse orrat MAO A or MAO B.

A deposit of the hybrid cell line identified herein as MAO-1C2 is ondeposit with the American Type Culture Collection and is assigned thenumber ATCC HB -8176.

The ability of MAO-1C2, coupled with secondary immunoglobulin reagents,to immunoprecipitate MAO B without inhibiting the enzyme or altering itssubstrate or inhibitor specificity makes MAO-1C2 monoclonal antibody anideal candidate as a sensitive test component for the detection of lowlevels of human MAO B.

E. Immunoassay using MAO-1C2 antibody for detection of MAO B in tissuesamples

All dilutions used RIA buffer (0.05 M TRIS buffer, pH 7.5, 0.14 M NaCl,10⁻³ M EDTA, 0.05% NP-40, 0.1% bovine serum albumin and 0.75%octylglucoside). Incubations were done in 96-well Cooke polystyrenemicrotiter plates at room temperature. Samples to be tested forcompetition were diluted to 100 μl with RIA buffer, and mixed with ³H-pargyline labeled, DEAE-purified platelet MAO (DEAE 7; 25 μl,containing 3.6 μg total protein and 30 ng of MAO, as measured by ³H-pargyline-binding capacity). A 1/2000 dilution of ammoniumsulfate-precipitated MAO-1C2 (25 μl) was added, and the microtiterplates were sealed with tape and incubated overnight with gentle rotaryshaking. Rabbit antimouse (IgG 10 μl, containing 40 μg of specificantibody) was added, and the incubation continued for 1 hr. Heat-killedfixed Staphylococcus aureus cells (50 μl of a 10% suspension, w/v; 24)were added, and after a further incubation for 15 min, the plates werecentrifuged for 15 min at 1700×g in an IEC centrifuge (model PR-6)equipped with Dynatech microplate carriers. Samples of the clearsupernatant (50 to 150 μl) were removed from each well, mixed withwater-miscible scintillation fluid and counted in a Packard TriCarbliquid scintillation spectrometer (counting efficiency, 54%).Calculation of RIA Data. Data was calculated with an Apple II Plusmicrocomputer equipped with Visicalc (Personal Software, Inc.,Sunnyvale, Calif.), programmed to calculate the % cpm remaining in thesupernatant and convert it to LN{(percent unbound)/(100-percentunbound)} (logit function). The logit function (dependent variable) forcompetition in the range of 25-70% was roughly linear when plottedversus log μg of protein added or log units of MAO B added. Estimates ofMAO B protein concentration were calculated by linear interpolation on astandard curve relating logit value to log μg MAO in DEAE-purifiedplatelet MAO. The total concentration of MAO B protein in the standardfraction was assumed to be equal to the concentration measured by ³H-pargyline binding (catalytically active enzyme).

Samples of extracts of gray matter, medulla, liver, and kidneycontaining 3-4 units of phenylethylamine (PEA)-oxidizing activity and oflung extract containing 0.44 units of MAO B activity were subjected toindirect immunoprecipitation radioimmunoassay using MAO-1C2, and theprecipitated and unprecipitated PEA-oxidizing activities weredetermined.

The results indicated that in all cases except lung, >90% of thePEA-oxidizing activity was found in the immunoprecipitates, unlessMAO-1C2 was omitted. Slightly less PEA-oxidizing activity (70%) wasprecipitated from the lung extract.

                  TABLE I                                                         ______________________________________                                        Specific concentration of MAO B in various tissues                            as measured by pargyline binding and radioimmunoassay.                                         Specific concentration                                                        (μg MAO/mg)                                                          Specific    Pargyline                                              Tissue     activity    binding  RIA                                           ______________________________________                                        Liver      225         12.4     10.9 ± 1.3                                 Lung       16.7        1.14     1.3 ± 0.0                                  Gray matter                                                                              34.2        1.38     2.8 ± 0.6                                  Medulla    62.2        1.91     2.1 ± 0.5                                  Kidney     205         9.2      12.2 ± 3.5                                 ______________________________________                                    

The data in Table I demonstrates that in all tissues except gray matter,there was excellent agreement between values of MAO proteinconcentration as measured by ³ H-pargyline binding assays (which measureonly catalytically active protein) and concentration measured by thecompetitive assay (hereafter termed the radioimmunoassay). Theradioimmunoassay appeared to detect about twice as much MAO protein inthe gray matter extract as the ³ H-pargyline binding assay. It ispossible that the gray matter extract studied here had higher levels ofcatalytically inactive but immunologically cross-reactive MAO moleculesthan the other extracts.

The results presented here, which are based on a study of theinteraction of MAO-1C2 with MAO B in various tissues from a singleindividual, suggest that MAO-1C2 may recognize human MAO B wherever itoccurs.

In another series of experiments using tissues from yet anotherindividual, it was found that MAO activity in extracts of mitochondriaof kidney and brain was immunoprecipitable with MAO-1C2 as expected.Immunoprecipitable MAO B activity has also been detected in diploidhuman skin fibroblasts, HeLa cells, and some clones of mouse-humanhybrid cells generated by the fusion of a diploid human skin fibroblastwith the mouse hepatoma cell line BWTG-3. Furthermore, quantitation bycompetitive radioimmunoassay of MAO B in individual platelet extractsfrom over 60 individuals (including both psychiatric and normalsubjects) that the MAO-1C2-defined determinant is associated withplatelet MAO B in all samples tested. Therefore, with the data nowavailable, it appears likely that the MAO-1C2-defined determinant isexpressed on all MAO B molecules in most tissues and all human subjects.

The foregoing description of the invention has been directed toparticular embodiments for purposes of explanation and illustration. Itwill be apparent, however, to those skilled in the art that manymodifications and changes in the processes of preparing and implementingthe described embodiments may be made without departing from the essenceof the invention. It is applicants' intention in the following claims tocover all equivalent modifications and variations as fall within thescope of the invention as defined by the following claims.

What is claimed is:
 1. A continuous monoclonal hybrid cell line which isclone MAO-1C2 identified as ATCC deposit HB-8176, developed as a fusionbetween a myeloma cell and a lymphocyte primed to human plateletmonoamine oxidase B purified to at least 20% by weight of total protein,which hybrid cell line is capable of producing antibody which reactswith human monoamine oxidase B enzyme and does not react with humanmonoamine oxidase A enzyme.
 2. A composition of matter consistingessentially of monoclonal antibody to human monoamine oxidase B enzymeproduced by hybridoma clone MAO-1C2 of claim 1 identified as ATCCdeposit HB-8176.
 3. An immunoassay method for the detection of monoamineoxidase B in a tissue sample, which method comprises exposing a tissuesample extract to a composition consisting essentially of monoclonalantibody to monoamine oxidase B enzyme produced from hybridoma cloneATCC deposit HB-8176 of claim 1, and subsequently determining the extentof monoamine oxidase B enzyme-antibody binding.